ABSTRACT MicroRNAs (miRNAs) are small non-coding RNAs approximately 21 nucleotides in length that regulate gene expression at the post-transcriptional level by inducing mRNA destabilization and translational inhibition of target mRNAs. In humans, more than 2000 miRNAs have been identified and are thought to control a variety of biological processes development and cancer. The goal of this competitive renewal application is to continue our investigation of the biological functions and the oncogenic properties of the polycistronic miR-17~92 miRNA cluster, also known as Oncomir1. The miR- 17~92 cluster contains six miRNAs (miR-17, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92) that can be grouped, based on sequence similarity, in four distinct subfamilies: miR-17 (includes miR-17 and miR-20a), miR-18, miR-19 (includes miR-19a and miR-19b), and miR-92a. During the past five years, supported by the NIH/NCI grant R01 CA149707, my group has performed a careful, and unprecedented, genetic dissection of this polycistronic cluster by generating an characterizing an allelic series of six genetically engineered mouse strains, each carrying selective targeted deletion of individual components of miR-17~92. This analysis, has lead to several important findings, including the discovery of causal role for miR-17 in skeletogenesis and axial patterning, the identification of germline mutations of miR- 17~92 as the cause of a human developmental syndrome known as Feingold Syndrome, and the discovery that the miR-19 family of miRNAs plays a key role in the pathogenesis of Myc-driven human cancers. These exciting new findings have lead to a series of novel hypotheses that are at the core of this grant renewal application. We propose a series of experiments that can be grouped into the following specific aims. The first aim has the goal of investigating the molecular mechanisms through which miR-19 contributes to Myc- driven tumorigenesis. We will also test the recently proposed hypothesis that miR-92 functionally antagonizes miR-19 by acting as a tumor suppressor. In the second aim, we will test a novel pharmacologic approach to inhibit miR-19 in vivo based on a recently developed technology that allows targeted delivery of miRNA antagonists to the acidic tumor microenvironment. In the third aim, we will develop a novel and more efficient method to experimentally identify miRNA-mRNA interactions directly in vivo. Successful completion of the experiments described in this grant application would greatly advance our understanding of this important miRNA cluster in particular, and of miRNAs in general, and could lead to novel anticancer approaches.